Tumor microenvironment (TME) makes an essential contribution to tumor progression, recurrence, and metastasis. Currently, there is no standardized method to quantify major components of TME simultaneously, such as lymphovascular invasion, and to predict locoregional recurrence and metastasis of cancer. The primary goal of this project is to develop a technology for quantification of the TME signatures by using quantum dot (QD)-based immunohistofluorescence (IHF). We expect that either single or multiplexed biomarkers for the TME components can be used to assess prognosis including risk of the locoregional recurrence and metastasis. We have recently developed QD-based IHF technology for quantification of multiplexed biomarkers. Our preliminary study support us to accomplish the objective of this study, that is, to develop a molecular imaging technology for quantification of major signatures of TME as a tool for assessment of the prognosis and prediction of the recurrence and metastasis. In this project, we will focus on technique development using head and neck cancer (HNC). It is anticipated that using this technology, HNC pathologists will be able to more accurately identify patients who are at high risk for poor prognosis. This would provide valuable information to head and neck cancer surgeons, radiation oncologist, and medical oncologists to make appropriate treatment decisions. This technology also has the potential to be applied to the treatment and care of other types of cancer with appropriate modifications. The project will involve 2 Specific Aims with 5 measurable milestones. Aim 1: To prepare and validate QD- antibody (Ab) conjugates which specifically recognize blood and lymphatic vasculatures, and EGFR in formalin-fixed and paraffin embedded (FFPE) cancer tissues. Milestones for this aim are (1) completion and optimization of 4 QD-Ab conjugates with high binding affinity and selectivity; (2) establishment of the quantification method for both density of the TME signatures and their distance to tumor nests; (3) validation of the QD-based IHF by comparing this method with the conventional IHC. Aim 2: To examine whether microvessel density and tumor proximity can serve as a prognostic biomarkers and correlate them with locoregional recurrence and lymph node metastasis of HNC. Milestones for this aim are (4) completion of staining and quantification of the TME signatures using 100 FFPE samples from HNC patients with and without lymph node metastasis and recurrent disease; (5) completion of statistical analysis to correlate microvessel density and tumor proximity with clinical characteristics of HNC patients.